Method of determining fatty acid composition of milk

ABSTRACT

A method of reducing the level of saturated fatty acids relative to the level of unsaturated fatty acids in milk. In particular, the genotyping and/or phenotyping of bovine cows on the basis of the amino acid residue located at position 67 of beta-casein produced in their milk. The invention is based on the finding that there is a correlation between the ratio of saturated to unsaturated fatty acids in milk and the beta-casein variants in milk.

TECHNICAL FIELD

This invention relates to a method for reducing the level of saturatedfatty acids relative to the level of unsaturated fatty acids in milk. Inparticular, the invention relates to the genotyping and/or phenotypingof bovine cows on the basis of the amino acid residue located atposition 67 of β-casein produced in their milk.

BACKGROUND

Dietary saturated fatty acids intake is known to be a major risk factorin heart disease in humans, particularly in countries where thepopulation is well-nourished. Animal products, such as dairy products(especially milk), are major contributors to the dietary intake ofhumans. It is generally accepted that the level of saturated fatty acidsfound in milk, particularly those with a chain length of less that 18carbon atoms, is a risk factor in coronary heart disease. In contrast,unsaturated fatty acids are considered to be beneficial. Because ofthis, there has been a preference for the consumption of plant derivedoils as opposed to animal based products.

The medical community is also concerned about the consumption of fatfound in milk because of the abundance of the saturated fatty acidC:14:0, which is thought to be atherogenic. The dairy industry has inpart responded with the production of “low fat” milk alternatives usingchemical separation and extraction techniques.

In addition to fats, specific protein components of milk, including theA¹ variant of the β-casein protein, are health risk factors. There are anumber of reports that the consumption of β-casein A¹ by humans islinked with a higher incidence of certain diseases, specificallydiabetes (Elliott et al. 1999 Diabetologia 42:292-6; Wasmuth et al. 1999Diabetologia 42 (Suppl. 1):A88 Proceedings of the Kongress derEuropäischen Diabetesgesellschaft vom 28.-30.09.1999 inBrüssels/Belgium) and coronary heart disease (McLachlan, C. N., Med.Hypotheses 56(2):262-72, 2001).

In addition to phenotyping a cow by identifying the particular β-caseinvariant or variants produced in the cow's milk, it is well known that acow can be genotyped for a specific single nucleotide polymorphism (SNP)to determine which β-casein variant or variants she will produce in hermilk. A method of selecting bovine cows on the basis of this genotypingmethodology to give milking herds which will produce milk free of theβ-casein A¹ variant, and preferably solely the β-casein A² variant, isthe subject of PCT/NZ96/00039 (published as WO 96/36239).

The applicant has now found that there is a correlation between theratio of saturated to unsaturated fatty acids in milk and the β-caseinvariants in milk. While there are known methods of altering the fattyacid composition of animal products, these typically include chemicalextraction, specific feeding and management systems, and quantitativegenetic selection for levels of specific fatty acids in milk. Eachmethod is costly and usually inefficient.

It is therefore an object of the invention to provide milk, or a productobtained from that milk, which has a reduced level of saturated fattyacids relative to unsaturated fatty acids, or to at least provide thepublic with a useful alternative.

STATEMENTS OF INVENTION

In a first aspect of the invention there is provided a method ofreducing the level of saturated fatty acids relative to the level ofunsaturated fatty acids in bovine milk by:

-   -   (a) determining which cows of a herd produce milk containing        β-casein having a proline at position 67, where the herd        comprises cows that produce milk containing β-casein having a        proline at position 67 and cows that produce milk β-casein        having a histidine at position 67, by testing genetic material        of individual cows of the herd for the presence of DNA encoding        β-casein having a proline residue at position 67 or by testing        milk produced by individual cows of the herd (or a product        produced from that milk) for the presence of β-casein having a        proline at position 67;    -   (b) selecting cows that have DNA encoding β-casein having a        proline residue at position 67 or cows that produce milk        containing β-casein having a proline at position 67; and    -   (c) milking the selected cows to give milk having a reduced        level of saturated fatty acids relative to the level of        unsaturated fatty acids compared with milk obtained from the        herd.

It is preferred that the β-casein having a proline at position 67includes one or more of β-caseins A², A³, D, E and F. It is alsopreferred that the β-casein having a histidine at position 67 includesone or more of β-caseins A¹, B, and C.

In a preferred embodiment of the invention the β-casein having a prolineat position 67 is β-casein A² and the β-casein having a histidine atposition 67 is β-casein A¹.

It is further preferred that, in addition to reducing the level ofsaturated fatty acids relative to the level of unsaturated fatty acidsin the milk produced by the herd of cows, the level of short and mediumchain saturated fatty acids having 6 to 14 carbon atoms in each chain(C6:0-C14:0) is also reduced.

In a further preferred embodiment of the invention, determining whichcows of the herd produce milk containing β-casein having a proline atposition 67 is by testing the genetic material of cows for the presenceof DNA encoding β-casein having a proline residue at position 67. In analternative embodiment, determining which cows of the herd produce milkcontaining β-casein having a proline at position 67 is by testing themilk produced by cows (or a product produced from that milk) for thepresence of β-casein having a proline at position 67.

While the genetic material of the cow may be any tissue containing, orwhich contained, nucleated cells, the genetic material is preferablyobtained from blood, hair, or milk.

In a second aspect of the invention there is provided milk obtained bythe method of the first aspect of the invention.

In a third aspect of the invention there is provided a milk productprepared from milk obtained by the method of the first aspect of theinvention.

In a fourth aspect of the invention there is provided a method ofaltering the proportions of saturated fatty acids and unsaturated fattyacids in a food by adding to the food an amount of β-casein having aproline at position 67.

Preferably the proportions of saturated fatty acids and unsaturatedfatty acids are altered by reducing the level of saturated fatty acidsin the food.

Preferably the food is milk or a milk product prepared from milk. It isalso preferred that the β-casein having a proline at position 67 isadded to the food by adding milk (or an extract from milk) obtained bythe method of the first aspect of the invention.

DETAILED DESCRIPTION OF THE INVENTION

It is well known that the genetics of an animal has a substantial impacton production levels and product quality, and on health, environmental,and animal welfare issues. The ability to determine a phenotype of ananimal by using a genetic test is a valuable tool for achieving rapididentification of animals and animal products with beneficialcharacteristics and for forming a group of animals having enhancedproduction and/or product quality. Animals can be grouped based ongenetic differences that relate to animal or animal product traits thatare of economic interest.

The gene (or variant of that gene) that is responsible for a particularphysical trait of an animal may, in some instances, be identifiable by asingle nucleotide polymorphism (SNP). An SNP is a DNA sequence at alocation in an animal's genome which is different to the DNA sequence atthe same location in the genome of another animal by virtue of only onenucleotide. Even a difference as small as this can mean one animalexhibits a particular physical trait whereas another animal does not.

Associations between the casein content and the fat content of milk havebeen identified, but these have been variable in the size and directionof the correlation. The results are therefore inconclusive. Bovenhiusand Weller (Genetics; 137(10):267-80, 1994) concluded that theassociations, where they exist, are due to linkage (in a sire pedigree)or linkage disequilibrium (in a population) with a fat QTL on the samecattle chromosome (chromosome 6). The overall conclusion from publisheddata is that the total amount of fat in milk is not related to β-caseingenotype and that the effect or effects that β-casein may have on humanhealth are not related to the volume of fat intake. However, theapplicant has now identified an unexpected relationship between thegenotype of the β-casein gene on cattle chromosome 6 and the fatty acidcomposition of cow milk.

The applicant has confirmed prior findings that milk which containsβcasein A¹ (A1 milk) has a similar overall percentage of fat comparedwith milk which is free of β-casein A¹ (A2 milk). Surprisingly, andcontradictory to previous findings, the applicant has discovered that A1milk has a higher percentage of saturated fatty acids and a lowerpercentage of unsaturated fatty acids compared to A2 milk. Alsosurprising was the finding that the levels of C6, C8, C10, C12 and C14fatty acids were reduced in milk from those cows homozygous for β-caseinA². This significant finding shows that milk which is substantially freeof β-casein A¹ will also produce milk fat that has lower levels ofsaturated fatty acids and medium chain fatty acids (C6 to C14) andhigher levels of unsaturated fatty acids. Therefore, this milk has thehealth benefit of the reduced risk of diseases associated with a highintake of saturated fatty acids, such as atherosclerosis, obesity,coronary heart disease, and diabetes.

Typically, a cow will produce β-caseins in its milk. However, differentβ-casein variants exist including A¹, A², A³, B, C, D, E, and F. Thedifferences between these proteins are determined by sequence variationsin the β-casein gene. For example, one difference is that the A², A³, D,E, and F variants have a proline residue at position 67 whereas the A¹,B, and C variants have a histidine residue at position 67. Thisdifference is determined by substitution of the nucleotide adenine withthe nucleotide cytosine at position 200 of the coding region of theβ-casein gene. The β-casein variant phenotype of a cow can be determinedindirectly by genotyping the SNPs that are responsible fordistinguishing these variant types.

The applicant has discovered that the selection of animals on the basisof β-casein variant type or the genetic variation in the β-casein genecan identify groups of animals with significant differences in theirmilk fatty acid compositions. For example, milk from animals which arehomozygous for the adenine nucleotide at position 200 of the codingregion of the β-casein gene (A1) differs in fatty acid composition frommilk from animals which are heterozygous for an adenine and cytosinenucleotide (A1/A2) at this position, which differs again from milk fromanimals which are homozygous for a cytosine at this position (A2).

More specifically, an adenine at position 200 of the β-casein geneincreases levels of the saturated fatty acids C6:0, C8:0, C10:0, C12:0and C14:0 and decreases unsaturated fat C18:1 by a comparable amount. Onremoval from consideration of the effects of herd, mob within herd,breed, age 2-8+, days in milk, methylation group, and sire, the β-caseingenotype accounts for 15-20% of the variation in these specific fattyacid profiles between animals.

The presence of a histidine at position 67 of β-casein enables theenzymatic formation of β-casomorphin-7. β-Casomorphin-7 is a seven aminoacid peptide that is formed only from β-caseins A¹, B and C. Casomorphinpeptides are known to act as opioids. Data from Lin et al. (1998,Peptides 19(2):325-31) suggest that β-casomorphin-7 may modulate theintake of dietary fat. β-Casomorphins stimulate the intake of dietaryfat in rats whereas enterostatin inhibits the intake. In addition, ithas been found that peptides from casein hydrolysates with tyrosyl endresidues (such as β-casomorphin-7) promote peroxidase-dependentoxidation of human LDLs (low density lipoproteins). Thus, the currentunderstanding of β-casein A¹, in terms of its relationship to factorsthat are detrimental to human health, is related to the action ofcasein, and peptides derived from it, on the fat metabolism of theconsumer and not related to differences in the fat composition of milkfrom animals of different β-casein genotype.

It is unlikely that the mechanism whereby β-casein affects the fattyacid composition of milk is due to a linked gene. This is because of thesize and consistency of the effect observed across sires. Withoutplacing any limitation on the invention, it is speculated that thediscovery is related to a direct effect of β-casein on the biosynthesisof lipids in mammary tissue. Alternatively, the discovery may be adirect result of the interactions of caseins with lipids in milk. If thelatter is correct, it may be possible to alter the fatty acid profile ofa product. Thus, the addition of β-casein obtained from animals ofselected casein variant type (for example, free of β-casein A¹) to aproduct under defined processing conditions may beneficially alter thefatty acid profile of the product.

The test for β-casein can be used to select animals to include in a herdfor milking or can be used to select animals to be used as sires, dams,or tissue donors for artificial breeding or cloning to breed subsequentgenerations of animals to be included in a herd for milking. In thisway, herds of milking cows can be formed which produce milk where theβ-casein A¹ protein is absent (or where the only β-casein present isβ-casein A²) in the protein fraction of the milk, and having reducedlevels of specific saturated fatty acids and increased levels of aspecific unsaturated fatty acids in the fat fraction of the milk. Amethod of selecting bovine cows on the basis of such genotyping to formmilking herds which will produce milk free of the β-casein A¹ variant,and preferably solely the β-casein A² variant, is the subject ofPCT/NZ96/00039 (published as WO 96/36239).

An additional feature of the invention is that once animals with aparticular genotype have been selected and milk is produced from them,the origin of the milk, or other products, such as milk powder andprocessed milk products, can be verified as being produced from theselected animals. This is achieved by determining the fatty acidcomposition of such a milk product. Consumers can therefore be confidentthat the milk is indeed from animals of the desired genotype.

The benefits of the milk of this invention are considerable:

-   -   (1) the absence of β-casein A¹ protein and the presence of only        β-casein A² produces a lower risk of coronary heart disease and        Type1 diabetes    -   (2) replacing saturated fat with unsaturated fat produces a        lower risk of coronary heart disease, obesity and other diseases    -   (3) the consumption of C14:0, which is thought to be        atherogenic, is reduced.

The mechanism by which casein effects the fatty acid composition of milkis unclear but it is possible that it is mediated though the formationof casomorphin peptides from casein. There may be a mechanisticrelationship between this and the effect of the consumption of β-caseinA¹ by humans. However, the direct effect of casein genotype on the fattyacid profile of milk has quite separate utility from the direct effectsof casein and casein metabolites on the metabolism of the consumer.There may also be a direct effect, whereby β-caseins (or particularvariants) can directly modify the fatty acid composition of milk.

EXAMPLES

DNA was extracted and the fatty acid compositions determined from milkfrom 1114 progeny derived from six sires which were heterozygous A/C atnucleotide 200 of the β-casein gene.

DNA was extracted from the milk in the following way. Milk was mixedthoroughly by inversion and 1.0 ml was pipetted into a 1.5 mlmicrocentrifuge tube. The tubes were centrifuged at 8,000 rpm for 10minutes and a 100 μl aliquot of supernatant (containing crude DNA)pipetted from each sample into a new 1.5 ml tube. The crude DNA extractwas stored frozen at −20° C. and 1-5 μl was used, without furtherpurification, for genotyping.

Genotyping methods used were have been described previously in detail inPCT/NZ96/00039 (published as WO 96/36239).

The samples for fatty acid analysis were centrifuged at 15,000 rpm for15 minutes. An aliquot of the upper layer of lipid was removed from eachsample. This lipid sample was heated to 60.0° C. and the melted lipidremoved, and stored frozen. The samples were subsequently methylated andanalysed by gas chromatography. The peak areas on chromatographs wereintegrated to quantify the levels each fatty acid. The identity of eachfatty acid was determined by comparing the retention time of each peakwith a known standard.

Of the samples analysed, animals either tested CC (A2), AC (A1/A2) or AA(A1) at position 200. The differences between genotypes were comparedusing generalised linear model analysis where the raw data was adjustedfor other factors which might affect fatty acid composition.Pre-adjustments were made for: Herd, Mob within Herd, Breed, Age 2-8+,Days in Milk, and Methylation Group within Herd. Finally, Sire,Genotype, and Sire by Genotype interaction were fitted.

The results from this study are given in Table 1 and show that the A2genotype had a significant effect on fatty acid composition. The levelsof statistical significance varied between individual fatty acids(*=p<0.05, **=p<0.1 ***=p<0.001). Compared to A1, milk from animals withthe A2 genotype had a significantly higher percentage of long chainunsaturated fatty acids (C18:1) and a lower percentage of saturatedmedium chain fatty acids in the range (C6:0-C14:0); whereas A1/A2individuals were intermediate for these values.

As a percentage of the total C18:1, A2-derived milk had about 3% moreC18:1 than A1-derived milk. C18:1 makes up about 15% of milk fat so theoverall effect as a proportion of total milk fat was about half apercent more C18:1. The reduction in the percentage of saturated fattyacids was similar to the increase in unsaturated fatty acid. With theeffects of herd, mob within herd, breed, age 2-8+, days in milk,methylation group and sire removed by the model, the β-casein genotypeaccounted for 15-20% of the variation in these specific fatty acidcompositions between the animals.

TABLE 1 Summary of β-casein genotype analyses for % fatty acids in milkfat Residuals for fatty acid traits (measured as % of total fatty acids)obtained from runs including all animals in the Ruakura Genetics trial.Pre-adjustments were made for: Herd, Mob within Herd, Breed, Age 2-8+,Days in Milk, Methylation Group within Herd C4:0 C6:0 C8:0 C10:0 C12:0C14:0 n = 3760 mean(%) 2.67 2.24 1.56 3.79 4.31 12.63  rsd(%) 0.29 0.190.12 0.42 0.52 0.81 adjR² 0.44 0.48 0.47 0.40 0.40 0.24 Model: Sire,β-casein genotype (A1; A1A2; A2), genotype × sire interaction. Traitsare standardised residuals. n = 1114 Sire ** * ** *** *** *** Genotypens * ** ** * ** Genotype × sire ns ns + ns ns ns Contrast A2-A1 −0.03 ±0.10 −0.25 ± 0.09 −0.29 ± 0.10 −0.26 ± 0.09 −0.21 ± 0.10 −0.24 ± 0.10(ns) (**) (**) (**) (*) (*) Contrast A2-A1A2 −0.13 ± 0.09 −0.16 ± 0.09−0.11 ± 0.09 −0.06 ± 0.09 −0.05 ± 0.09 −0.03 ± 0.09 (ns) (+) (ns) (ns)(ns) (ns) Contrast A1-A1A2 −0.10 ± 0.08  0.09 ± 0.07  0.18 ± 0.07  0.20± 0.07  0.16 ± 0.07  0.20 ± 0.07 (ns) (ns) (*) (**) (*) (**) C18:1 C15:0C16:0 C16:1 C18:0 C18:1 trans C18:2 CLA 1.36 30.48  1.33 9.45 15.69 3.48 1.36 0.84 0.16 2.71 0.29 1.37 1.85 0.87 0.20 0.27 0.26 0.22 0.260.28 0.27 0.34 0.60 0.36 Model: Sire, β-casein genotype (A1; A1A2; A2),genotype × sire interaction. Traits are standardised residuals. *** ****** *** *** *** *** *** ns ns ns ns * ns ns ns ns ns ns ns ns ns ns ns 0.01 ± 0.10 −0.07 ± 0.10  0.04 ± 0.10 0.01 ± 0.09 0.26 ± 0.10 0.08 ±0.10  0.09 ± 0.11 0.16 ± 0.10 (ns) (ns) (ns) (ns) (**) (ns) (ns) (ns)−0.01 ± 0.09 −0.09 ± 0.09 −0.02 ± 0.09 0.09 ± 0.09 0.12 ± 0.09 0.07 ±0.10 0.001 ± 0.10 0.10 ± 0.09 (ns) (ns) (ns) (ns) (ns) (ns) (ns) (ns)−0.02 ± 0.08 −0.02 ± 0.08 −0.06 ± 0.08 0.08 ± 0.07 −0.14 ± 0.07  −0.003± 0.08  −0.09 ± 0.08 −0.06 ± 0.08  (ns) (ns) (ns) (ns) (+) (ns) (ns)(ns)

Although the invention has been described by way of example, it shouldbe appreciated that variations and modifications may be made withoutdeparting from the scope of the invention. Furthermore, where knownequivalents exist to specific features, such equivalents areincorporated as if specifically referred in this specification.

INDUSTRIAL APPLICABILITY

Milk having a low level of saturated fatty acids compared to unsaturatedfatty acids is useful for the avoidance of certain diseases anddisorders. Dietary fatty acid intake is a major risk factor in heartdisease and much of that dietary fatty acid intake is from theconsumption of milk and milk products. The ability to obtain milk low insaturated fatty acids relative to unsaturated fatty acids by milkingonly those cows that have been genotyped or phenotyped on the basis oftheir ability to produce β-casein variants having proline, rather thanhistidine, at position 67 represents a useful method of producing milkbeneficial to human health.

1. A method of determining relative levels of saturated fatty acids andunsaturated fatty acids in bovine milk obtained from one or more cows:(a) testing genetic material of the one or more cows for the presence ofDNA encoding β-casein having a proline residue at position 67 or DNAencoding β-casein having a histidine residue at position 67; (b)identifying whether a cow or cows (i) will likely have a lowerpercentage of saturated fatty acids and a higher percentage ofunsaturated fatty acids based on being homozygous for DNA encodingβ-casein having a proline residue at position 67 or (ii) will likelyhave a higher percentage of saturated fatty acids and a lower percentageof unsaturated fatty acids based on being homozygous for the DNAencoding β-casein having a histidine residue at position 67 for whichgenetic material was tested in (a); and (c) obtaining milk from at leastone of the one or more cows after step (b).
 2. The method as claimed inclaim 1, wherein the β-casein having a proline at position 67 includesone or more of β-caseins A2, A3, D, E and F.
 3. The method as claimed inclaim 2, wherein the β-casein having a proline at position 67 isβ-casein A2.
 4. The method as claimed in claim 1, wherein the β-caseinhaving a histidine at position 67 includes one or more of β-caseins A1,B, and C.
 5. The method as claimed in claim 4, wherein, the β-caseinhaving a histidine at position 67 is β-casein A1.
 6. The method asclaimed in claim 1, wherein the genetic material of the cows may be anytissue containing, or which contained, nucleated cells.
 7. The method asclaimed in claim 6, wherein the genetic material is obtained from blood,hair, or milk.